Continuous flow zonal rotor

ABSTRACT

A method and apparatus for continuous flow centrifugation wherein a sample is introduced through the top surface of a rotor core into a sample zone adjacent a gradient zone so that the sample passes substantially 360* and a sample gradient is formed in said gradient zone. The spent sample is removed through the bottom surface of said rotor core, and the sample gradient is removed after separation by introducing a liquid gradient through the top surface of the rotor core to the gradient zone to displace the sample gradient radially inward. The sample gradient is passed out of the core while the rotor is spinning through a passage at the core axis at a reduced pressure in the passage. Since only one liquid stream enters or exits at or near the core axis, the possibility of sample product contamination is eliminated.

nite States Patent [1 1 Joyce 51 July 24, 1973 CONTINUOUS FLOW ZONALROTOR [75] Inventor: John E. Joyce, South Weymouth,

Mass.

[73] Assignee: Damon Corporation, Needham,

Mass.

[22] Filed: Apr. 9, 1971 [211 App]. No.: 132,663

[52 us. Cl 2213/32, 2233/16, 233 1 D [51] Int. Cl. 1304b 1/00 [58] Fieldof Search 233/27, 28, l R, 233/1 D, 32, 33, 46, 47 R, 16, 17, 21

[56] References Cited UNITED STATES PATENTS 3,536,253 10/1970 Anderson233/33 3,430,849 3/1969 Gibson et a1 233/33 X 3,168,474 2/l965 Stallmanet al. 233/33 3,073,517 1/1963 Pickels et al....' 233/32 1,795,9583/1931 McFarlane 233/28 3,291,387 12/1966 Billen 233/28 3/1970 Harbott233/16 3,519,201 7/1970 Eisel et al. 233/21 Primary Examiner-George H.Krizmanich Attorney-Kenway, Jenney & Hildreth [57] ABSTRACT A method andapparatus for continuous flow centrifugation wherein a sample isintroduced through the top surface of a rotor core into a sample zoneadjacent a gradient zone so that the sample passes substantially 360 anda sample gradient is formed in said gradient zone. The spent sample isremoved through the bottom surface of said rotor core, and the samplegradient is removed after separation by introducing a liquid gradientthrough thetop surface of the rotor core to the gradient zone todisplace the sample gradient radially inward. The sample gradient ispassed out of the core while the rotor is spinning through a passage atthe core axis at a reduced pressure in the passage. Since only oneliquid stream enters or exits at or near the core axis, the possibilityof sample product contamination is eliminated.

11 Claims, 7 Drawing Figures PATENIEUJUtZdIBH SHEU 1 OF 3 INVENTOR JOHNE. JOYCE wmzm ATTORNEYS CONTINUOUS FLOW ZONAL ROTOR BACKGROUND OF THEINVENTION This invention relates to continuous flow centrifugation andparticularly to a method and apparatus for recovering sample gradientsfrom continuous flow centrifugal rotors.

In continuous flow centrifugation, a sample to be separated is pumpedcontinuously into a rotating centrifuge and into a liquid gradient zone.The gradient zone is formed so that the liquid gradient density, whichincreases with its radial position away from the rotor center, isregulated to separate sample strata therein. By regulating the liquidgradient density to accommodate the density of the liquid sample, sampleseparation can be effected at any desired radial position. Generally thedesired radial position is chosen for ease of sample recovery.

While the centrifuge is rotating, the gradient liquid remains stationaryin the radially outward region or zone, during the treatment of itssample. The density gradient of the gradient liquid usually is aconcentration gradient of a relatively non-diffusing suitable solutesuch as salt or sucrose. The sample liquid is pumped continuously into azone radially intermediate the gradient liquid and the rotor and iscaused to circulate around all or a portion of the rotor circumference.The gradient functions to entrap particles sedimenting from the sampleliquid without further radial movement of the particles at that locationwhere their density equals that of the gradient. The travel of particlesof greater or lesser density is varied accordingly. As a consequence,there will be in the gradient, particles separated radially asindividual populations of different densities that may be recoveredseparately after the sample flow has been terminated and while the rotoris spinning. Even in the case of particles that cannot be suspendedwithin the gradient, it functions to so curb their radial velocity that,depending on the flow period, their impingement with the outer boundaryof the rotor chamber is prevented even though no effective purificationis accomplished.

Some zonal rotors have separate sectors and the sample is divided intostreams flowing separately through its sectors. Such sectors are ofrelatively large volume, each sector typically representing 25 percentof the capacity of the rotor. In addition, a zonal rotor is availablehaving a particle collecting sector that extends substantially 360 topermit the sample to travel the same distance within the. rotor. Thelatter centrifuge is preferable since it provides more effective sampleseparation. This centrifuge is described in a copending application toJohn E. Joyce entitled "Rotors and Rotor Cores for Continuous FlowCentrifuges", Ser. No. 129,055 filed Mar. 29, 1971.

In present continuous flow zonal centrifuges, major problems areassociated with the sample recovery step. Presently, the sample to beseparated is introduced and the recovered sample is removed adjacent therotor axis and appropriate sealing is effected to prevent mixing of thetwo. However, these sealing arrangements have proven unreliable underthe forces encountered during centrifugation and undesirable mixture ofthe incoming and recovery streams is commonly encountered.

ln present systems when a sample or gradient liquid is being introducedinto the spinning rotor, either during sample separation or samplerecovery, a liquid in the rotor is displaced and is removed therefromand the liquid must be introduced at a pressure above atmosphericwhether or not the liquid is being introduced at a radially inward oroutward position with respect to the liquid being removed. Theseincreased liquid pressures place additional stress on the seals betweenthe liquid streams thereby making it essential that the sealing surfacesbe machined to almost perfect flatness, usually to within 50 toAngstroms. These machine tolerances are difficult to attain therebymaking present systems undesirable from the standpoint of highmanufacturing expense as well as unreliability.

It would be highly desirable to provide a centrifuge method andapparatus for continuous flow centrifugation wherein the possibility ofleakage between liquid streams is eliminated. Furthermore, it would bedesirable to provide an inexpensive sealing arrangement which permitsliquid recovery from a spinning centrifuge that does not requiremachining to extremely close tolerances.

SUMMARY OF THE INVENTION The present invention provides a method andapparatus for continuous flow centrifugation of samples and in oneaspect provides a novel sealing arrangement. A centrifuge rotor isprovided with two circular grooves in its top surface, each groove beingadjacent the other and radially separated from each other. First andsecond passages through the rotor, connect the grooves with either asample zone or a gradient zone located between the peripheral rotorsurface and the housing. The first passages have an outlet radiallyoutward from the second passage outlets. A third passage has an inlet atsample zone and an outlet in the bottom surface of the rotor to removethe spent sample. A fourth passage connects the sample zone with anoutlet conduit at the rotor axis which can be connected with a means forreducing pressure below atmospheric. The fourth passage has a seal witha rotating section and a stationary section that permits sample removalfrom the spinning rotor. The reduced pressure employed is defined by theequation:

wherein d(lbs/cu. in.) is the density of the fluid being removed, N(rpm)is the speed of the rotor, r(in) is the radial position of sample drainoutlet and B is a proportionality constant equal to 2.841 X 10"".

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more fullydescribed with reference to the accompanying drawings.

FIG. 1 is a top view of a centrifuge rotor.

FIG. 2 is an elevation cross-sectional view of the apparatusfor'removing separated sample.

FIG. 3 is a partial cross-section view taken along line 3 of FIG. 1.

FIG. 4 is a cross-sectional view taken along line 4 of FIG. I.

FIG. 5 is a cross-sectional view taken along line 5 of FIG. 1.

FIG. 6 is a partial cross-sectional view taken along Line 6 of FIG. 1.

FIG. 7 is a partial cross-sectional view taken along Line 7 of FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIGS. 1 and 3through 7, the centrifuge generally indicated at 1 comprises a rotorhousing 2 and a rotor 3 having a frusto-conical shape. The rotor housing2 has a lower axial socket 8 to receive a motor drive (not shown) and anupper axial socket 9 to receive a sealed sample recovery systemdescribed hereafter with reference to FIG. 2. The rotor 3 is providedwith a plurality of fluid passages for the introduction and exit ofliquid gradient and fluid sample in a continuous cycle so that thesample travels about 360 around the rotor 3. The large arrows indicatethe direction and path of sample flow. The liquid gradient andintroduction of sample is accomplished as follows: While the rotor 3 isspinning, liquid gradient is introduced into circular groove 10 byplacing the outlet of a tube adjacent to or into the groove 10 fordelivery of fluid thereto. The liquid gradient passes from groove 10through a plurality of passageways 11 that terminate at the ends of fins12a, 12b, 12c and 22 which are positioned adjacent the rotor housing 2.Introduction of liquid gradient into groove 10 and passageways 11 iscontinued until the desired size of the gradient zone 13A is effected.Generally, the gradient zone 13A extends from the inner surface of rotorhousing 2 to the radially outermost point of rotor 3. After the gradientzone 13A has been established, sample to be separated is introduced intocircular groove 15 which is radially inward from groove 10 and isconnected with sample delivery passage 16 having an outlet 17 that ispositioned radially inward from the gradient zone 13A. The fluid sampleis constantly fed to groove 15 and into passageway 16 so that there isestablished inside the rotor 3, while it is spinning, a sample zone 18that travels clockwise around the rotor 3 substantially 360 until itreaches drain passageway 19 and outlet 20 through which spent sample isremoved from the rotor 3.

While the sample zone 18 travels around the rotor, liquid gradient zone13A does not move relative thereto while it is spinning. Bypass ofsample around fin 22 is prevented by means of circular plate 23 andbottom plate 24 which are adhered to rotor housing 2 and prevent passageof sample over or under the fin 22. Plates 23 and 24 extend thecircumference of rotor housing 2 and plate 24 is provided with anopening to allow spent sample to drain (See FIG. 6). The sample zone 18passes through opening 21 of fins 12 but does not bypass fin 22 sincefin 22 does not have an opening through which the sample gradient canpass. Accordingly, the sample must pass out of outlet 19 and drain 20.While the sample passes through zone 18, particles therein separatetherefrom to form strata in the liquid gradient 13A. After the desiredamount of sample has passed through the rotor 1, sample delivery ingroove 13A and separated sample particles therein toward the inlet 25 ofsample removal passageway 26. Prior to introducing the liquid gradientafter sample separation and during the introduction of the new liquidgradient, the sealing arrangement shown in FIG. 2 is positioned so thatfluid passing from passage 26 through outlet 27 can pass into inlet 28of a sample removal tube 29. The sealing arrangement shown in FIG. 2comprises generally two sections; a rotating seal 30 and a stationaryseal 31. The rotating seal 30 and stationary seal 31 are maintained inclose contact by means of spring 32. The rotating seal housing 33 isheld in the main seal housing 34 by means of ball bearings 35 and theseal housing 33 is moved into and out of rotor 3 by means of lever 36which is pivoted around pin 37 and bears against the top surface 38 ofseal housing 34. The lever 36 moves against the action of spring 40.When positioned to remove sample, the extension 30a sealed to rotatingseal 30 fits into an arbor 44 which in turn, is fit into socket 9 andsealed therein by means of O ring 45. The extension 30a is sealed inarbor 44 by means of O ring 46. When the rotor housing 2 is rotated,arbor 44, extension 30a, seal 30 seal housing 33 hearing 35 also rotatewhile seal 31, spring 32, housing 34, tube 29, spring 40 and lever 36 donot rotate. The main seal housing 34.

is fixed to a housing for the centrifuge (not shown) by means of a sealshelf 42 and a safety disc 43 attached to shelf 42. The plate 42 isprovided with openings 10a and 15a to accommodate filling tubes that canextend into the grooves 10 and 15. Sample recovery is effected by meansof pump 50 which reduces the pressure in collection chamber 51; tube 29,and passage 26 when valve 52 is open and valves 53 and 54 are closedthereby causing gradual withdrawal of the liquid gradient and the samplestrata therein while liquid gradient is being introduced into passage11, as described above. Sample recovery is effected 'while the rotorhousing 2 is spinning at moderate speeds usually about 1000 to 2000 rpmso that the sample strata remain separate in the liquid gradient duringrecovery. The sample strata are recovered sequentially, with the leastdense stratium being recovered first. After the desired sample portionis collected in collection chamber 51, valve 52 is closed and valves 53and 54 are open so that the sample can be recovered through conduit 55.

The drain outlet for spent sample is radially outward from the radiallyouter most groove to obtain the desired liquid flow. It is preferredthat the groove adapted to receive liquid gradient during samplerecovery be located radially outward from the groove adapted to receivethe sample to attain desired liquid flow. Also, it is preferred that thegrooves be inclined or shaped so the lower portion thereof extendsradially further outward than the upper portion to reduce spillage fromthe grooves onto the top surface of the rotor. It is preferred that theoutlet for the liquid gradient passage extend to the inner surface ofthe housing to provide ease in gradient zone formation and separatedsample removal. in this respect, the liquid gradient passage need notextend below the openings 21 in fins 12a, 12b and 12c. A

tube through the openings 21 can be employed to connect liquid gradientpassages in the rotor body and the radially outermost portion of thef'ins.

While the invention has been described above with reference to a rotorwherein sample travels about 360 and wherein four sample passages andfour liquid gradient inlet passages are employed, it is to be understoodthat variations in the apparatus can be made to attain the same results.Thus, the rotor can be segmented by closing the openings 21 in fins 12a,12b and 120 and additional sample inlet passages and drains are providedfor each rotor segment so that the sample travels only about 90 throughthe rotor. However, this embodiment is not preferred since sampleseparation therein is less complete for a given sample flow rate.Furthermore, more or less sample outlet and gradient inlet passages canbe employed to attain equivalent sample separation and recovery.Furthermore, any sealing arrangement can be employed so long as it iseffective in providing passage for one liquid stream and to retainadequate sealing between mating rotatable and stationary sections.

I claim:

1. A method for forming a sample gradient which comprises forming agradient zone in a spinning centrifuge, flowing a sample through the topsurface of a rotor core into a sample zone adjacent the gradient zone sothat the sample passes substantially 360 and a sample gradient is formedin said gradient zone, removing spent sample through the bottom surfaceof said rotor core, and removing the sample gradient by introducingliquid gradient through the top surface of the rotor core to thegradient zone to displace said sample gradient radially inward andpassing said sample gradient out of said core while the rotor isspinning through a passage at the axis of the core at a subatmosphericpressure in said passage.

2. A continuous flow centrifuge rotor comprising a rotor core havingmeans for introducing two separate streams of liquid through circulargrooves in the top surface of the rotor so that one stream can bedirected into a sample zone and the other stream can be directed into agradient zone, a means for removing spent sample from said sample zonethrough an outlet on the bottom surface of said rotor and means forremoving separated sample from said rotor through a passage at the axisof said rotor while the rotor is spinning.

3. The centrifuge of claim 2 having means for flowing a sample streamthrough said sample zone substantially 360 around said rotor core.

4. A continuous flow centrifuge comprising a rotor housing a rotor corewithin said housing and spaced from said housing to define a gradientzone adjacent said housing and a sample zone between said rotor and saidgradient zone, said rotor core having a top surface, a bottom surfaceand a peripheral surface, said top surface having'a first circulargroove and a second circular groove radially spaced from said firstcircular groove each groove adapted to receive means for delivering aliquid, at least onepassage connecting said first groove with saidgradient zone andat least one passage connecting said second groove withsaid sample zone at least one drain passage in said core connecting saidsample zone and said bottom surface, said drain passage having an outletradially intermediate the radially outermost groove and the peripheralsurface, at least one sample recovery passage in said core connectingthe sample zone and an axial passage at the axis of said core and saidaxial passage having means to recover separated sample at subatmosphericpressure in said axial passage pressure while said rotor is spinning.

5. The centrifuge of claim 4 having means for flowing a sample streamthrough said sample zone substantially 360 around said rotor core.

6. The centrifuge of claim 5, four passages; equally radially spaced fordelivery gradient liquid to said gradient zone and four sample recoverypassages equally radially spaced.

7. The centrifuge of claim 5 wherein a plurality of first fins extendfrom said rotor core into the gradient zone adjacent said housing, eachof said first fins having an opening to permit the sample zone to passtherethrough and a solid second fin extending from said rotor core intothe gradient zone adjacent the housing a sample stream inlet and asample stream outlet being positioned adjacent opposing verticalsurfaces of said second fin and plate means adhered to the bottom andtop surfaces of the second fin extending from the gradient zone to therotor core to prevent by-pass of said sample zone around said secondfin.

8. The centrifuge of claim 6 wherein a plurality of first fins extendfrom said rotor core into the gradient zone adjacent said housing, eachof said first fins having an opening to permit the sample zone to passtherethrough and a solid second fin extending from said rotor core intothe gradient zone adjacent the housing, a sample stream inlet and asample stream outlet being positioned adjacent opposing verticalsurfaces of said second fin and plate means adhered to the bottom andtop surfaces of the second fin extending from the gradient zone to therotor core to prevent by-pass of said sample zone around said secondfin.

9. The centrifuge of claim 7 .wherein the passages connecting the firstgroove with the gradient zone extend through the second fin and througheach opening in the first fins.

10. The centrifuge of claim -8 wherein the passages connecting the firstgroove with the gradient zone extend through the second fin and througheach opening in the first fins.

11. A method for forming a sample gradient which comprises forming agradient zone in a spinning centrifuge, flowing a sample through the topsurface of a rotor core into a sample zone adjacent to the gradient zoneso that the sample passes substantially 360 and a sample gradient isformed in said gradient zone, removing spent sample through the bottomsurface of said rotor core, and removing the sample gradient byintroducing liquid gradient through the top surface of the rotor core tothe gradient zone to displace said sample gradient radially inward andpassing said sample gradient out of said core while the rotor isspinning through a passage at the axis of the core.

4 t t it t UNITED STATES PATENT oFHcE CERTIFICATE OF CORECHUN Patent'NQ,3,747,843 Dated July 24, 1973 Inventor(s) JOHN E JOYCE It is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Column 6, line S, cancel "pressure" Signed and sealed this 22nd day ofJanuary 1974..

(S-EAL) Attest: 1

EDWARD M.FLETCHER,JR. RENE D. TEGTMEYER Attesting Officer ActingCommissioner of Patents

1. A method for forming a sample gradient which comprises forming agradient zone in a spinning centrifuge, flowing a sample through the topsurface of a rotor core into a sample zone adjacent the gradient zone sothat the sample passes substantially 360* and a sample gradient isformed in said gradient zone, removing spent sample through the bottomsurface of said rotor core, and removing the sample gradient byintroducing liquid gradient through the top surface of the rotor core tothe gradient zone to displace said sample gradient radially inward andpassing said sample gradient out of said core while the rotor isspinning through a passage at the axis of the core at a subatmosphericpressure in said passage.
 2. A continuous flow centrifuge rotorcomprising a rotor core having means for introducing two separatestreams of liquid through circular grooves in the top surface of therotor so that one stream can be directed into a sample zone and theother stream can be directed into a gradient zone, a means for removingspent sample from said sample zone through an outlet on the bottomsurface of said rotor and means for removing separated sample from saidrotor through a passage at the axis of said rotor while the rotor isspinning.
 3. The centrifuge of claim 2 having means for flowing a samplestream through said sample zone substantially 360* around said rotorcore.
 4. A continuous flow centrifuge comprising a rotor housing a rotorcore within said housing and spaced from said housing to define agradient zone adjacent said housing and a sample zone between said rotorand said gradient zone, said rotor core having a top surface, a bottomsurface and a peripheral surface, said top surface having a firstcircular groove and a second circular groove radially spaced from saidfirst circular groove each groove adapted to receive means fordelivering a liquid, at least one passage connecting said first groovewith said gradient zone and at least one passage connecting said secondgroove with said sample zone at least one drain passage in said coreconnecting said sample zone and said bottom surface, said drain passagehaving an outlet radially intermediate the radially outermost groove andthe peripheral surface, at least one sample recovery passage in saidcore connecting the sample zone and an axial passage at the axis of saidcore and said axial passage having means to recover separated sample atsubatmospheric pressure in said axial passage pressure while said rotoris spinning.
 5. The centrifuge of claim 4 having means for flowing asample stream through said sample zone substantially 360* around saidrotor core.
 6. The centrifuge of claim 5, four passages; equallyradially spaced for delivery gradient liquid to said gradient zone andfour sample recovery passages equally radially spaced.
 7. The centrifugeof claim 5 wherein a plurality of first fins extend from said rotor coreinto the gradient zone adjacent said housing, each of said first finshaving an opening to permit the sample zone to pass therethrough and asolid second fin extending from said rotor core into the gradient zoneadjacent the housing a sample stream inlet and a sample stream outletbeing positioned adjacent opposing vertical surfaces of said second finand plate means adhered to the bottom and top surfaces of the second finextending from the gradient zone to the rotor core to prevent by-pass ofsaid sample zone around said second fin.
 8. The centrifuge of claim 6wherein a plurality of first fins extend from said rotor core into thegradient zone adjacent said housing, each of said first fins having anopening to permit the sample zone to pass therethrough and a solidsecond fin extending from said rotor core into the gradient zoneadjacent the housing, a sample stream inlet and a sample stream outletbeing positioned adjacent opposing vertical surfaces of said secOnd finand plate means adhered to the bottom and top surfaces of the second finextending from the gradient zone to the rotor core to prevent by-pass ofsaid sample zone around said second fin.
 9. The centrifuge of claim 7wherein the passages connecting the first groove with the gradient zoneextend through the second fin and through each opening in the firstfins.
 10. The centrifuge of claim 8 wherein the passages connecting thefirst groove with the gradient zone extend through the second fin andthrough each opening in the first fins.
 11. A method for forming asample gradient which comprises forming a gradient zone in a spinningcentrifuge, flowing a sample through the top surface of a rotor coreinto a sample zone adjacent to the gradient zone so that the samplepasses substantially 360* and a sample gradient is formed in saidgradient zone, removing spent sample through the bottom surface of saidrotor core, and removing the sample gradient by introducing liquidgradient through the top surface of the rotor core to the gradient zoneto displace said sample gradient radially inward and passing said samplegradient out of said core while the rotor is spinning through a passageat the axis of the core.